Investigation of the effect of Myricetin on Cisplatin-induced liver hepatotoxicity

SUMMARY OBJECTIVE: Cisplatin, a widely used anticancer agent, induces hepatotoxicity alongside organ damage. Understanding Cisplatin's toxicity mechanism and developing preventive measures are crucial. Our study explores Myricetin, a flavonoid, for its protective effects against Cisplatin-induced hepatotoxicity. METHODS: In our study, a total of 32 Wistar albino male rats were utilized, which were categorized into four distinct groups: Control, Myricetin, Cisplatin, and Myricetin+Cisplatin. For the histological assessment of hepatic tissues, hematoxylin–eosin and periodic acid Schiff staining were employed, alongside immunohistochemical measurements of TNF-α, interleukin-17, and interleukin-6 immunoreactivity. Additionally, aspartate transaminase and alanine transaminase values were examined by biochemical analysis. RESULTS: In the histological evaluation of the tissues, a normal healthy cell structure and a strong periodic acid Schiff (+) reaction were observed in the hepatocyte cells in the tissues of the Control and Myricetin groups, while intense eosinophilia, minimal vacuolization, congestion, and sinusoidal expansions were observed in the hematoxylin–eosin stainings, and a decrease in the positive reaction in the periodic acid Schiff staining was observed in the Cisplatin group. Consistent with these histological findings, an increase in TNF-α, interleukin-17, and interleukin-6 expressions (p<0.0001) and a concomitant increase in aspartate transaminase and alanine transaminase values were observed in the Cisplatin group. In the group protected by Myricetin, a significant improvement was observed in all these histological and biochemical values. CONCLUSION: Cisplatin induces notable histopathological alterations in the liver. In this context, Myricetin exhibits the potential to alleviate Cisplatin-induced damage by modulating histological parameters and biochemical processes.


Experimental design
The rats were randomly assigned to four groups of eight.Control group: rats that had access only to water and food throughout the experiment; Cis group: a single dose (7.5 mg/kg) of Cis was administered intraperitoneally on the seventh day 10 ; Myr group: Myr (10 mg/kg) was administered intraperitoneally for 7 days 11 ; Myr+Cis group: Myr (10 mg/kg) was administered intraperitoneally for 7 days, and at the end of the seventh day, a single intraperitoneal dose of Cis (7.5 mg/kg) was given.After the experimental procedure, the rats were anesthetized and then sacrificed.

Chemicals
Cisplatin (Koçak Farma, Istanbul, Turkey) was used intraperitoneally as an inducer of liver damage.Myr (Sigma-Aldrich, St. Gallen, Switzerland) was used as a protective and therapeutic substance in the experiment.

Histological examination
At the end of the experiment, rats were anesthetized using anesthetic agents [ketamine (75 mg/kg)+xylazine (10 mg/kg)].Liver tissues were fixed in a 4% formaldehyde solution.Then, the routine light microscopic procedure was applied.For this procedure, dehydration was first applied to the tissues.Then, it was made transparent by holding it in xylene, and fixed blocks were made with paraffin.Sections were taken from paraffin blocks and stained with hematoxylin-eosin and periodic acid-Schiff (PAS).Sections were examined under a light microscope 12 .
To determine the changes occurring as a result of damage to the liver tissue, the immunohistochemical staining method was applied to show the expressions of TNF-α, IL-17, and IL-6 12 .

Biochemical analysis
Alanine aminotransferase and AST values of blood serum samples taken at the end of the experiment were analyzed by the service in the Erciyes University Central Biochemistry Laboratory.

Histopathological findings
The histological structure of normal healthy cells was observed in the liver sections of the control and Myr groups.It is seen that some of the hepatocytes in the Cis-treated group have more intense eosinophilic staining.It is seen that there is irregularity in the arrangement of the cell cords and widening and distortions in the sinusoidal spaces in some sections.However, areas of congestion and mononuclear cell infiltration were detected in the tissues belonging to the damage group.In addition, in the liver tissues of the Myr+Cis-applied group, there was a decrease in eosinophilic staining compared to the damage group, the hepatocyte arrangement around the central vein was more regular, and the widening in the sinusoidal spaces decreased (Figure 1).
Periodic acid Schiff staining was performed to evaluate the glycogen content of liver tissues.In the sections of the control and Myr groups, it was observed that hepatocyte cells gave a strong PAS-positive reaction.However, in the Cis-applied group, there was a decrease in PAS positivity compared to the control group.In addition, an increase in PAS positivity density was observed in the Myr+Cis-applied group (Figure 1).

Immunohistochemical findings
In the study, immunohistochemical staining was performed to determine the TNF-α, IL-17, and IL-6 immunoreactivity of the experimental groups.When TNF-α protein expression was examined, a significant increase was observed in the Cis group applied alone compared to the other groups, while this increase was observed to be minimally reduced in Cis applied together with Myr.Similarly, a significant increase in IL-17 and IL-6 expression was observed in the Cis group administered alone, while a statistically significant improvement was observed in the Cis group administered together with Myr (Figure 2 and Table 1).
Aksoy S et al.

Biochemical fındings
While minimal changes were observed between the groups in AST values, in the comparison of the Cis group applied alone and the Cis group applied together with Myr in ALT values, it was seen that Myr corrected the increase in the damage group statistically significantly (Table 1).

Statistical analyses
In the study, statistical analysis of the results obtained from biochemical and immunoreactivity data was performed using GraphPad (Prism 8.00 for Mac, GraphPad Software, La Jolla, California, USA).The D'Agostino Pearson omnibus test was used to check the normal distribution of the data.Data were expressed as mean±SD and analyzed by one-way ANOVA test and Tukey's post-hoc test for parametric tests.p<0.05 was considered significant in the analysis.

DISCUSSION
It is known that Cis causes damage to many tissues, and one of these negative effects is liver hepatotoxicity 13,14 .Cis causes morphological changes in the arrangement of hepatocyte cords 15 .For example, in the liver, irregularity in the hepatic cords, portal triad fusion and central vein obstruction 16 , degenerative hepatocytes 14 , pyknosis of hepatocyte nuclei around the vena centralis in some and hypertrophy, inflammation, hypertrophy in some hepatocytes, vascular occlusion, sinusoidal dilatation 17 , and congestions are a few of them 18 .
Similar to the results of these studies, according to our histological data, in the liver sections of rats administered Cis alone, compared to the control group, there were changes in the classical lobule structure, intense eosinophilia in hepatocytes, thickening of the vena centralis wall, minimal vacuolar changes in the cytoplasm, sparse mononuclear cell infiltration, and enlargements of the sinusoids.It was determined that the strong PAS-positive reaction seen in the control group decreased in the damage group.This decrease in PAS positivity may be due to damaged mitochondria and decreased glucose levels.Histological data of the Cis group applied together with Myr show that cellular deteriorations were improved and there was an increase in the PAS-positive reaction in hepatocytes compared to the damage group.After Cis administration, significant glycogen loss is observed in hepatocytes.Myr prevents this glycogen loss.Glycogen positivity in hepatocytes is confirmed by amylase incubation, which abrogates the PAS reaction in these compartments 15 .
Single-dose Cis administration increases AST, ALT, and ALP activities 19 and causes a significant increase in serum TNF-α levels compared to the control group 16,20 .Our data in our study increased the serum AST and ALT values of the group administered a single dose of Cis, similar to the literature.However, a significant improvement was observed in liver enzyme values, especially ALT values, in the group protected by Myr against Cis-induced damage.
Similarly, in Cis-induced damage studies, severe TNF-α expressions in the Cis group 3 and an increase in oxidant parameters, a decrease in antioxidant parameters, and a severe increase in TNF-α and Caspase-3 expressions in immunohistochemical evaluations were noted 21 .Our findings in our study are similarly manifested by the upregulation of TNFα, IL-17, and IL-6 in the damage group.In hepatotoxicity, Myr prevents hepatotoxicity by modulating the production of free radicals and inflammatory markers.Additionally, Myr treatment reduced hepatotoxicity and ethanol-induced inflammatory markers such as IL-6 6 .Apart from this, hemorrhagic necrosis of liver tissues in hepatotoxicity and inflammatory cell infiltration in the portal area were significantly reduced by Myr pretreatment, resulting in less bleeding and cell infiltration, indicating that Myr has a protective effect on liver There is no significant difference between groups containing the same letter (a-d).p<0.05 was considered significant.
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tissues 22 .In our current study, although the histological disorders and biochemical changes occurring in the injury group showed a partial improvement in TNF-α expression in the immunohistochemical values of the Myr group applied for protective purposes along with Cis, a significant improvement was observed in IL-17 and IL-6 protein expressions.Biochemical values similarly support these findings.

CONCLUSION
The decrease in histological damage markers and biochemical activities of Myr against Cis-induced hepatotoxicity unequivocally demonstrates its protective effect on cellular structure, highlighting the need to enhance the dose and duration of Myr application to optimize its effectiveness, which constitutes a crucial avenue for further research.

Table 1 .
Liver tissue TNF-α, interleukin-17, and interleukin-6 immunoreactivity measurement results and serum aspartate transaminase and alanine transaminase results of the experimental groups.